This chapter provides a broad overview of many applications of plasmids for genetic analysis, primarily in bacteria. Ever since DNA sequencing became accessible to most research laboratories, reverse genetic analysis has become a standard experimental approach to study bacterial gene function. Similar suicide vectors have also been used for nontargeted insertional mutagenesis by cloning random chromosomal DNA fragments into the plasmid. The use of suicide vectors also allows for easy identification of the insertion mutations. Plasmids that utilize different combinations of double-counter selective markers have been used for diverse applications, including the search for extremely rare suppressor mutations of essential Escherichia coli genes, and to improve the efficiency of allelic exchange on bacterial artificial chromosomes (BACs). Although temperature-sensitive vectors represent the majority of conditionally replicating plasmids, other plasmids that exhibit conditional replication have been described. Cloning by recombination was also achieved using the highly efficient DNA uptake and recombination systems in Acinetobacter calcoaceticus. Site-specific recombination machinery has also been incorporated into several expression vector systems to achieve very tight regulation of gene expression. Although antibiotic resistance is typically used to maintain selection for plasmids grown in culture, there are disadvantages to the use of antimicrobial agents for certain industrial, medical, and biotechnological applications.

Allelic exchange mutagenesis using a temperature-sensitive replicon. The top portion of the figure shows the composition of a circular plasmid with a temperature-sensitive ori (orirs). A mutant allele is created in vitro by insertion of an antibiotic-resistance gene (abr1) into a gene targeted for mutagenesis (a continuous line with arrowhead indicates an intact gene and a broken line represents an interrupted gene; P and t represent promoter and terminator, respectively). The allele may be a simple insertion (as shown) or an insertion/deletion mutation. The temperature-sensitive vector also carries a distinct antibiotic-resistance marker (abr2) and may also include a counterselection marker such sacB. Integration of the vector into the host chromosome occurs at the nonpermissive temperature by homologous recombination (crossover event at position A). The configuration of the integrated plasmid is shown in the middle, showing the duplication of the targeted sequences. Growth at the permissive temperature for plasmid replication selects for transformants that have undergone a second recombination event at position A or B. Only recombination at position B results in allelic exchange. Counterselection, such as growth on sucrose, may also be used to enhance the frequency at which recombinants that have undergone exchange at position B are recovered. The configuration of the chromosome after allelic exchange is shown at the bottom. The insertion mutation has been recombined onto the chromosome, and the wild-type allele is now carried by the vector and can complement the insertion mutation at the permissive temperature. If counterselection is used, however, the vector will be lost from the cell.

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10.1128/9781555817732/fig28-1.gif

Figure 1

Allelic exchange mutagenesis using a temperature-sensitive replicon. The top portion of the figure shows the composition of a circular plasmid with a temperature-sensitive ori (orirs). A mutant allele is created in vitro by insertion of an antibiotic-resistance gene (abr1) into a gene targeted for mutagenesis (a continuous line with arrowhead indicates an intact gene and a broken line represents an interrupted gene; P and t represent promoter and terminator, respectively). The allele may be a simple insertion (as shown) or an insertion/deletion mutation. The temperature-sensitive vector also carries a distinct antibiotic-resistance marker (abr2) and may also include a counterselection marker such sacB. Integration of the vector into the host chromosome occurs at the nonpermissive temperature by homologous recombination (crossover event at position A). The configuration of the integrated plasmid is shown in the middle, showing the duplication of the targeted sequences. Growth at the permissive temperature for plasmid replication selects for transformants that have undergone a second recombination event at position A or B. Only recombination at position B results in allelic exchange. Counterselection, such as growth on sucrose, may also be used to enhance the frequency at which recombinants that have undergone exchange at position B are recovered. The configuration of the chromosome after allelic exchange is shown at the bottom. The insertion mutation has been recombined onto the chromosome, and the wild-type allele is now carried by the vector and can complement the insertion mutation at the permissive temperature. If counterselection is used, however, the vector will be lost from the cell.

Knockout mutagenesis by use of an R6Kγori suicide vector. A suicide vector is constructed by inserting a DNA fragment representing an internal portion of the target gene (the region of homology between the target gene and region carried by the suicide vector is shown by diagonal crosshatching). The vector is introduced to the host by transformation or conjugation with selection for the antibiotic-resistance marker (abr). Recombination between homologous sequences results in disruption of the target gene, as shown by the bottom portion of the figure (a continuous line with arrowhead indicates an intact gene and a broken line represents an interrupted gene; P and t, represent promoter and terminator, respectively).

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Figure 2

Knockout mutagenesis by use of an R6Kγori suicide vector. A suicide vector is constructed by inserting a DNA fragment representing an internal portion of the target gene (the region of homology between the target gene and region carried by the suicide vector is shown by diagonal crosshatching). The vector is introduced to the host by transformation or conjugation with selection for the antibiotic-resistance marker (abr). Recombination between homologous sequences results in disruption of the target gene, as shown by the bottom portion of the figure (a continuous line with arrowhead indicates an intact gene and a broken line represents an interrupted gene; P and t, represent promoter and terminator, respectively).

Allelic exchange mutagenesis by using an R6Kγori suicide vector and counterselection. The top portion shows an R6Kγori suicide vector carrying a DNA fragment representing an internal portion of the target gene (diagonal crosshatching) and disrupted with an antibiotic-resistance marker (abr1). The vector also carries a counterselection marker, e.g., sacB and a distinct antibiotic-resistance marker (abr2). As described in the text, other counterselection markers may also be used. Recombination between homologous sequences (crossover event at position A) results in disruption of the target gene (a continuous line with arrowhead indicates an intact gene and a broken line represents an interrupted gene; P and t represent promoter and terminator, respectively) with duplication of the homologous sequences. Growth in the presence of appropriate antibiotic and sucrose (for sacB) selects for recombinants that have undergone a second recombination event at position B. The configuration of the chromosome after allelic exchange and loss of the suicide vector is shown at the bottom.

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10.1128/9781555817732/fig28-3.gif

Figure 3

Allelic exchange mutagenesis by using an R6Kγori suicide vector and counterselection. The top portion shows an R6Kγori suicide vector carrying a DNA fragment representing an internal portion of the target gene (diagonal crosshatching) and disrupted with an antibiotic-resistance marker (abr1). The vector also carries a counterselection marker, e.g., sacB and a distinct antibiotic-resistance marker (abr2). As described in the text, other counterselection markers may also be used. Recombination between homologous sequences (crossover event at position A) results in disruption of the target gene (a continuous line with arrowhead indicates an intact gene and a broken line represents an interrupted gene; P and t represent promoter and terminator, respectively) with duplication of the homologous sequences. Growth in the presence of appropriate antibiotic and sucrose (for sacB) selects for recombinants that have undergone a second recombination event at position B. The configuration of the chromosome after allelic exchange and loss of the suicide vector is shown at the bottom.

Construction of unmarked mutations by recombineering. Two strategies for engineering unmarked deletion/ insertion and point mutations into the bacteria chromosome or BACs using linear DNA recombination are shown. On the left, PCR is used to amplify an antibiotic resistance (abr) cassette using primers (short arrows) whose 5' ends (gray extensions) are homologous to the gene targeted for mutagenesis (line with arrowhead flanked by P and t, representing promoter and terminator, respectively). The abr cassette is flanked by frt or lox site-specific recombinase recognition sites (arrowheads). The right portion of the figure shows similar primers used to amplify a cassette containing both a selectable marker (abr) and a counterselection gene (sacB). The middle portion of the figure depicts the events after the linear DNA fragments are introduced by electroporation into an E. coli strain expressing bacteriophage recombinases (Red or RecET) with selection for antibiotic resistance. After replacement of the target gene, abr can be eliminated by expression of the appropriate site-specific recombinase, e.g., Flp, leaving only a short "scar" sequence (single arrowhead), as shown on the bottom, left. Alternatively, a second round of recombineering can be performed using a linear DNA fragment to which a desired point mutation or deletion (white box) has been introduced. Recombinants are recovered by selecting against the counterselection marker.

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Figure 4

Construction of unmarked mutations by recombineering. Two strategies for engineering unmarked deletion/ insertion and point mutations into the bacteria chromosome or BACs using linear DNA recombination are shown. On the left, PCR is used to amplify an antibiotic resistance (abr) cassette using primers (short arrows) whose 5' ends (gray extensions) are homologous to the gene targeted for mutagenesis (line with arrowhead flanked by P and t, representing promoter and terminator, respectively). The abr cassette is flanked by frt or lox site-specific recombinase recognition sites (arrowheads). The right portion of the figure shows similar primers used to amplify a cassette containing both a selectable marker (abr) and a counterselection gene (sacB). The middle portion of the figure depicts the events after the linear DNA fragments are introduced by electroporation into an E. coli strain expressing bacteriophage recombinases (Red or RecET) with selection for antibiotic resistance. After replacement of the target gene, abr can be eliminated by expression of the appropriate site-specific recombinase, e.g., Flp, leaving only a short "scar" sequence (single arrowhead), as shown on the bottom, left. Alternatively, a second round of recombineering can be performed using a linear DNA fragment to which a desired point mutation or deletion (white box) has been introduced. Recombinants are recovered by selecting against the counterselection marker.

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